1. Field of the Invention
The present invention generally relates to radioactive staining of gels to identify proteins. More specifically, the invention relates to the preparation of a radioactive stain composition, and to the utilization of the radioactive stain composition in an overall method of identifying proteins.
2. Description of the Prior Art
The arsenal of stains available for the detection of polypeptides in polyacrylamide gels after separation by electrophoresis is already quite extensive but there is always a quest for more sensitive reagents. The enhancement of detection that is afforded by recently developed techniques, such as the silver stain technique, not only compensates for the diminished protein load in methods employing very thin gels but can also reveal polypeptide components that are undetected by conventional Coomassie Blue stain. See article by C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert in Science, vol. 211, pp. 1437-1438 (1981).
One of the most powerful visualization tools, radioautography, is presently limited to experiments which are conducted with intrinsically labelled proteins produced from substrates containing radioactive labelled amino acids. See article by P. H. O'Farrell, in J. Biol. Chem., vol. 250, pp. 4007-4021 (1975). Moreover, extrinsic radioactive labelling techniques, such as iodination, can alter the electrophoresis properties of proteins, and the isotope is not necessarily uniformly linked to each protein and non-selectively distributed among all polypeptide components of the mixture.
Direct iodination of separated proteins in polyacrylamide gels has been reported by Edler et al (see the article by J. H. Edler, R. A. Peckett II, J. Hampton, and R. A. Lerner in the J. Biol. Chem., vol. 252, pp. 6510-6515 (1977)). This technique is quite useful when used to study radioactive peptides after tryptic digestion, but it has been found that some lots of acrylamide contain a contaminant which also becomes radioiodinated, and this negates its utility for radioautography.
It has been reported that ferrous bathophenanthroline sulfonate (BPS) is well suited for protein staining because of its reproducability. When so utilized, it rapidly stains and destains, yielding clear backgrounds and reproducable results. See the article by G. Graham, R. S. Nairn and G. W. Bates in Anal. Biochem., vol. 88, pp. 434-441 (1978).
However, such prior art techniques are burdened by certain disadvantages. For example, such techniques are often quite complex and slow. In addition, such prior art techniques often involve modifications of the proteins prior to electrophoresis.
Furthermore, in prior art techniques involving radioiodination of proteins, the radioiodination procedure may be hazardous to the user. In those techniques involving prior labelling, such prior labelling requires individual treatment (experimentation) for each sample. In addition, the prior art techniques, by virtue of their complexity and slowness, are necessarily quite costly.
Finally, prior art techniques are typically not sensitive to low-level protein presence; that is, such techniques are not as sensitive as they should be, and accordingly cannot demonstrate low protein levels. In the techniques of the prior art, such as those involving sensitive stains such as silver, such sensitive stains do not stain all proteins with equal efficiency; accordingly, a non-uniformity in staining results.